| Objective:The individual can make quick reaction once suffering harmful stimulation and set up a stable recognition of the information about this so that when the similar situation reappears,he would make the right reaction to protect himself,which is a beneficial mechanism for survival.A random harmful stimulation has its own mechanism for information establishing or degrading,both of which could make profound impacts on the individual.It involves many different brain areas to dispose associative information.Hippocampus plays important role in recognizing the context.Amygdala is significant in the procession of aversive stimulation,along with obvious emotion reaction.Additionally,integration of associated-information is closely related with prefrontal cortex(PFC),which may decide the final response to the stimulation.The rodent like living in groups and they have the steady social behavioral system which is important for foraging,mating and escaping since they need a protection for the continuation and development of their race.The males are more territorial and they would become extremely aggressive when other types of mice intrude their territory and attack them.So,we utilize this kind of trait to establish the social defeat model.This research utilizes social attack to make the social defeat model,observing the individual's reaction to the harmful stimulation,the time tendencies of memory establishment and extinction and the reaction of amygdala-hippocampus-PFC.Methods:1.Setting up social defeat model: Experimental C57BL/6(body weight,20±2g)mice were exposed to a novel Kunming(body weight,30±3g)aggressor for 10 min,and then separated from the aggressor behind a protective barrier,which was perforated to allow for sensory contact,after 6 hours,taking out protective barrier and repeating social defeat.When the second attack finished,C57BL/6 mice were placed back into their home cage.These animals were randomly assigned into control group(n = 10)and social defeat groups.Rats in social defeat groups were divided into subgroups according to the time points at which animals were sacrificed(1d,3d,7d and 15d;n = 10 per subgroup)2.Behavioral test: including Social Interaction Test,Open-field Test(OFT)and Sucrose Preference Test at 1day,3days,7days and 15 days after the social defeat.3.Tissue preparation and procedure of assay: under anesthesia(3.5% chloral hydrate,0.1ml/10g),blood was collected from orbit in 1.5ml micro-tubes.Afterwards,blood samples were centrifuged(8000 rpm,10 min,4°C)and plasma was stored at-20°C until assayed.Plasma samples were analyzed using commercially available ELISA kit for ACTH and GC.At corresponding time points,5 mice from each group were perfused with 4% paraformaldehyde,and then the brain was harvested to make the coronal sections(30?m)by freezing microtome and the coronal sections were harvested for immunohistochemistry for c-Fos in discrete brain region.5 mice from each group were decapitated then collected the bilateral hippocampus,amygdale and PFC tissues,frozen and stored at-80?.Unilateral regions of brain were used to extract total RNA being used for reverse transcription into cDNA or to extract total protein.Using Real-time PCR to test the transcription of zif268(egr1)and Arc and using Western Blot to test c-fos level in hippocampus,amygdala and PFC.Results:1.Behavioral changes and expression of IEGs: after social defeat,the moving distance and moving time were obviously decreased and social interaction behavior was reduced too compared with the control group.At the same time,after 1day and 3days,in the amygdala,Arc and zif268 levels increased significantly,but in hippocampus and PFC deceased and the numbers of c-fos positive cells increased in amygdala and decreased in ventral-hippocampus and PFC;the protein level of c-fos had the same tendency of IHC.7 days and 15 days after social defeat stimulation,the moving time,moving distance and the social interaction gradually increased;the Arc and zif268 levels increased in both hippocampus and amygdala,but PFC still has a low levels for these two IEGs;the number of c-fos positive cells decreased in amygdala,but in ventral-hippocampus and PFC increased;the protein level of c-fos was still maintain highly in amygdala and increased in both of hippocampus and PFC.When it comes to Sucrose Preference Test,the experimental group shows reduction of sucrose water.2.Hormone change: by testing with ELISA,plasma GC level considerably increased after stimulation and sustained for 8hours.Then,the GC and ACTH levels were decreased at 1 day and3 days and gradually regained.The GR and FKBP5 protein levels in hippocampus increased after1 day and 3 days,then decreased in 7 days and 15 days.The mRNA expression of FKBP5 has the similar tendency with protein,but the m RNA expression of GR decreased after social defeat.Conclusion:Acute social defeat reduced the motivation and social interaction of mice in the open field.The behavioral changes were obvious at 1 day and 3 days after social defeat,which indicates that aggressive stimulation has been established.Between 3 days and 7 days after the stimulation,the time of exploration raised.After 7days,the time of social interaction raised which means the reduction of harmful information.Our results showed that the characteristic of time course is closely related to the reaction of neuron in hippocampus,amygdala and PFC.Particularly,the aversive memory establishment and memory consolidation are correlatively related with continuous excitation of amygdala.After the social defeat,the reaction of neuron in hippocampus(ventral hippocampus)decreased,which means low-response when contacts with the similar scene.The exploration following aversive stimulation is mainly interrelated to restrain of PFC.Following the aversive information's extinction,the excitation of neuron in amygdala is reduced,and in hippocampus and in PFC,this excitation is increased being accompanied by much more exploration.The direct or indirect anatomy connections between amygdala,hippocampus(ventral-hippocampus)and PFC were provided by AAV9-GFP injection.The social defeat stimulation could cause the stress reaction and we suggests the secretions of GC and ACTH into circulation also have the time tendency,and this stimulation could also cause the GR-FKBP5 signal change in hippocampus.In conclusion,we have identified this kind of memory resulting from the social defeat has the particular tends for establishment and extinction.The harmful information maintains least7 days and then gradually degrades.The establishment and consolidation of harmful memory is commonly related with amygdala,meanwhile,the harmful stimulation could restrain the acceptance of new information in hippocampus and PFC.For the harmful information fading away,the reactions of neuron decrease in amygdala and increase in hippocampus and PFC.And the GC level in circulation expresses the similar time tendency to the neuron reaction. |
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